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Antimicrobial peptide MP1102 of anti-drug resistance staphylococcus aureus and preparation method and application thereof

A technology of MP1102 and A-MP1102, applied in botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of high production cost, insufficient antibacterial activity, and low yield of gene cloning expression

Active Publication Date: 2013-09-25
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antimicrobial peptides have a wide antibacterial spectrum, poor stability, cytotoxicity, insufficient antibacterial activity, drug diversity, high production costs (gene cloning expression yield is too low and chemical synthesis cost is too high), etc. have always been faced in the application of antimicrobial peptides Challenge (Eckert, 2011)

Method used

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  • Antimicrobial peptide MP1102 of anti-drug resistance staphylococcus aureus and preparation method and application thereof
  • Antimicrobial peptide MP1102 of anti-drug resistance staphylococcus aureus and preparation method and application thereof
  • Antimicrobial peptide MP1102 of anti-drug resistance staphylococcus aureus and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Antimicrobial Peptide MP1102 Sequence Design

[0061] The physical and chemical parameters of NZ2114 series derivative peptides were statistically analyzed by bioinformatics tools, and the structural parameters of NZ2114 were optimized in combination with the structure and activity of antimicrobial peptides to reduce the instability coefficient and fat cluster coefficient of NZ2114, increase the hydrophobic moment of NZ2114, and increase α According to the helicity parameters, the 9th, 13th, and 14th amino acids of NZ2114 were subjected to site-directed mutations to design a new antimicrobial peptide MP1102. The primary amino acid sequence of MP1102 was: GFGCNGPWQEDDVKCHNHCKSIKGYKGGYCAKGGFVCKCY.

[0062] The primary sequence result parameters are shown in Table 2:

[0063] Table 2 Analysis of structural parameters of antimicrobial peptides

[0064]

[0065] The secondary structure was analyzed by bioinformatics software (http: / / emboss.bioinformatics.nl / ), ...

Embodiment 2

[0066] Example 2 Acquisition of the gene encoding the antimicrobial peptide MP1102

[0067] (1) Optimization of the MP1102 gene expression sequence encoding the antimicrobial peptide MP1102

[0068] According to the yeast codon table Pichia pastoris[gbpln]:137CDS's(81301codons)

[0069]

[0070]

[0071]

[0072] The DNA sequence after codon optimization is shown in SEQ ID No:2. Sequence feature: 120bp; type: nucleic acid; chain type: double-stranded; topology: linear; molecular type: double-stranded DNA.

[0073] (2) Design of gene expression cassette

[0074] Based on effectively terminating the translational expression of MP1102, two consecutive TAA stop codons were inserted at the C-terminus of the MP1102 coding sequence. The signal peptide cleavage site kex2 site was inserted into the N-terminus of MP1102 to realize the natural secretion and expression of MP1102 in Pichia pastoris. XhoI was designed at both ends of the MP1102 gene, XbaI endonuclease sites Xho...

Embodiment 3

[0091] Example 3 Recombinant Yeast Expression Vector Construction

[0092] (1) The MP1102 gene obtained in Example 2 and the pPICZαA vector were double digested with XhoI and XbaI endonucleases.

[0093] The double enzyme digestion system is as follows:

[0094]

[0095] Digestion conditions: 37°C, 4h in water bath.

[0096] The digested product was recovered with a DNA product recovery kit (purchased from Tiangen Biological Co., Ltd.), and stored at -20°C for future use.

[0097] After the MP1102 gene and the pPICZαA vector were digested with XbaI and XhoI, the MP1102 gene was connected to the linearized pPICZαA vector with T4 DNA ligase. The connection system was as follows:

[0098]

[0099] Connection conditions: 25°C, 1h.

[0100] (2) Transform the recombinant vector in (1) into Escherichia coli DH5α, and the specific operation steps are as follows: Freeze and store Escherichia coli DH5α competent cells at -80°C, and thaw immediately on ice. Take 90 μL of compet...

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Abstract

The invention provides a novel antimicrobial peptide MP1102 of anti-drug resistance staphylococcus aureus, which has strong bactericidal activity on the staphylococcus aureus including MRSA. An encoding gene of the antimicrobial peptide MP1102 is optimized to create a pPICZalphaA-MP1102 recombinant expression vector, pichia pastoris X-33 is linearly converted by BglII to obtain recombinant yeast strain M5 with high level expression of MP1102 so as to realize the high level secretory expression of MP1102 in the pichia pastoris X-33 and create an M5 transformant high density fermentation system, and as shown in detection results of total protein concentration, antimicrobial peptide MP1102 concentration and biomass, the total protein concentration reaches 809mg / L after being fermented at 25 DEG C for 120 hours, the antimicrobial peptide MP1102 concentration reaches 420mg / L, the antimicrobial peptide MP1102 accounts for 52% of the total protein, and the biomass reaches 353g / L. The MP1102 has the potential of being developed as a new generation of antibacterial agents.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a novel antimicrobial peptide MP1102 against drug-resistant Staphylococcus aureus, its preparation method and application. Background technique [0002] Antimicrobial peptides (AMPs) are small molecule polypeptides with biological activity in organisms. The main characteristics are as follows: high antibacterial activity, wide antibacterial spectrum, and many types, and they have killing or inhibiting effects on fungi, protozoa, even viruses and tumor cells (Brandenburg, et al, 2012). Due to its outstanding biological characteristics, it has the most potential as a new type of safe and harmless antibiotic substitute, and thus has attracted the attention of the scientific community (Brandenburg, et al, 2012; Eckert, 2011). [0003] The application of antimicrobial peptides instead of traditional antibiotics in various diseases caused by bacterial infections has always att...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/81C12N1/19A61K38/17A61P31/04C12R1/84
Inventor 王建华毛若雨滕达王秀敏张勇
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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